Genetic determinants and absence of breast cancer in Xavante Indians in Sangradouro Reserve, Brazil.

Genetic compositions of distinct human populations are different. How genomic variants influence many common and rare genetic diseases is always of great medical and anthropological interest, and understanding of genetic architectures of population groups in relation to diseases can advance our knowledge of medicine. Here, we have studied the genomic architecture of a group of Xavante Indians, an indigenous population in Brazil, and compared them with normal populations from the 1000 Genomes Projects. Principal component analysis (PCA) indicates that the Xavante Indians are genetically distinctive when compared to other ethnic groups. No incidence of breast cancer cases has ever been reported in the population, and polygenic risk analysis indicates extremely low breast cancer risk in this population when compared with germline TCGA (The Cancer Genome Atlas) breast cancer normal control samples. Low germinal mutation burden among this population is also observed. Our findings will help to deepen the understanding of breast cancer and might also provide new approaches to study the disease.

Ulnar-Sided Wrist Pain in the Athlete: Sport-Specific Demands, Clinical Presentation, and Management Options.

ABSTRACT: Ulnar-sided wrist injuries are common in sports that require repeated pronosupination, wrist radial/ulnar deviation, axial loading, and gripping equipment. Common anatomic structures affected include the triangular fibrocartilage complex, extensor carpi ulnaris tendon, distal radioulnar and ulnocarpal joints, and hamate bone. Presenting symptoms include pain with activity, swelling, possible snapping or clicking, and reproduction of symptoms with provocative maneuvers. Imaging may confirm or rule out pathologies, but abnormal findings also may present in asymptomatic athletes. Initial treatment is usually nonoperative with splinting, load management, activity modification, strengthening the components of the kinetic chain of the particular sport, and pain management. Surgery is usually indicated in ulnar-wrist pain pathology such as hook of hamate fractures and required in associated instability. Future research should address specific treatment and rehabilitation protocols, emphasizing the complete kinetic chain along with the injured wrist.

Hypermethylation of the progesterone receptor A in constitutive antiprogestin-resistant mouse mammary carcinomas.

Most breast carcinomas that are estrogen receptor (ER) and progesterone receptor (PR) positive respond initially to an endocrine therapy, but over time, they develop resistance (acquired hormone resistance). Others, however, fail to respond from the beginning (constitutive resistance). Overcoming hormone resistance is one of the major desirable aims in breast cancer treatment. Using the medroxyprogesterone acetate (MPA)-induced breast cancer mouse model, we have previously demonstrated that antiprogestin-responsive tumors show a higher expression level of PR isoform A (PRA) than PR isoform B (PRB), while tumors with constitutive or acquired resistance show a higher expression level of PRB. The aim of this study was to investigate whether PRA silencing in resistant tumors was due to PRA methylation. The CpG islands located in the PRA promoter and the first exon were studied by methylation-specific PCR (MSP) in six different tumors: two antiprogestin-responsive, two constitutive-resistant, and two with acquired resistance. Only in constitutive-resistant tumors, PRA expression was silenced by DNA methylation. Next, we evaluated the effect of a demethylating agent, 5-aza-2'-deoxycytidine, on PRA expression and antiprogestin responsiveness. In constitutive-resistant tumors, 5-aza-2'-deoxycytidine treatment in vitro and in vivo restored PRA expression and antiprogestin RU-486 responsiveness. Furthermore, high levels of DNA methyltransferase (Dnmts) 1 and 3b were detected in these tumors. In conclusion, our results suggest that methyltransferase inhibitors in combination with antiprogestins may be effective in the treatment of constitutive-resistant carcinomas with a high DNA methyltransferase level.

Entropy of Feulgen-stained 17-beta-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis.

MCF-10F human breast epithelial cells when transformed with 17-beta-estradiol (E2) give rise to highly invasive C5 cells that generate adenocarcinomas in SCID mice. From these tumors, cell lines such as C5-A6-T6 and C5-A8-T8 have been derived. Variable patterns of chromatin supraorganization have been demonstrated for these cells during the transformation/tumorigenesis progress, when assessing chromatin entropy by image analysis in Feulgen-stained preparations. Since epigenetic dysregulation might contribute to the chromatin textural repatterning in transformed MCF-10F cells, the association of the variable chromatin packing states with global DNA methylation was investigated in these cells after their treatment with restriction enzymes followed by Feulgen staining and chromatin entropy evaluation by image analysis. The results indicate that although -CmCGG- sequences may affect chromatin supraorganization in some of the analyzed cell types (perhaps due to localized hypermethylation), not all the chromatin condensation patterns in these cells with transformation and/or tumorigenesis are associated with DNA methylation (e.g. E2 cells). Chromatin supraorganization remodeling in C5-A6-T6 and C5-A8-T8 cells may be attained by different mechanisms, with C5-A6-T6 chromatin packing states perhaps being associated with local DNA hypermethylation or other epigenetic factors, and C5-A8-T8 likely being associated with global DNA hypomethylation, as reported in the literature for other cell types. Thus, we assume that a variable epigenetic modulation affecting the higher-order packing states of chromatin in the estrogen-transformed MCF-10F cell model could be evident with the chromatin entropy study by image analysis.

Image analysis of the AgNOR response in ras-transformed human breast epithelial cells.

The argyrophylic staining of the nucleolar organizer regions (AgNOR positive response) in interphase nuclei is often related directly to the cellular demand for ribosome biogenesis and is considered of relevance in studies of tumor pathology. Transformation of human breast epithelial MCF-10A cells by the c-Ha-ras oncogene results in altered growth, invasiveness and tumorigenicity in nude mice. Since ras transformation may be associated with a more intense nucleolar activity, we examined the influence of transfection by the Ha-ras oncogene on AgNOR staining response in MCF-10A cells. Following assessment of the AgNOR response with video image analysis, the AgNOR-positive areas and the AgNOR area/nuclear area ratio, but not the number of AgNOR aggregates or dots per nucleus, were found to be much higher after ras transformation. A role of the Ha-ras transformation on the nucleolar activity of the MCF-10A is thus suggested as assessed by the AgNOR staining. Based on data in the literature, it is also hypothesized that a decreased wild-type p53 level, possibly promoted by the ras transformation, may be associated with the increased AgNOR response.

17-beta-estradiol affects nuclear image properties in MCF-10F human breast epithelial cells with tumorigenesis.

Treatment of the human breast epithelial cells MCF-10F with 17-beta-estradiol (E2) induces transformation and tumorigenesis. E2-transformed MCF-10F cells are known to exhibit progressive loss of ductulogenesis, and invasive and tumorigenic phenotypes. Although DNA amounts and chromatin supraorganization change in E2-transformed MCF cells, no comparative study has yet been undertaken in the resulting cells selected for aggressive invasiveness (C5) and tumor generation in a heterologous host. The aim of this study was thus to determine whether changes in Feulgen-DNA content and chromatin supraorganization are involved during E2-induced transformation and tumorigenesis of the MCF-10F cells. Image analysis was performed for nontransformed and E2-transformed MCF cells, highly invasive cells (C5), and for cell lines (C5-A6-T6 and C5-A8-T8) derived from tumors generated by injection of C5 cells in SCID mice. A decrease in Feulgen-DNA amounts and nuclear sizes induced by E2 treatment was accented with selection of the highly invasive tumorigenesis potential. However, in the tumor-derived cells a high variability in cellular phenotypes resulted inclusively in near-polyploidy. Significant changes in textural parameters, including nuclear entropy, indicated chromatin structural remodeling with advancing tumorigenesis. An increased variability in the degree of chromatin packing states in the E2-transformed MCF cells is followed by reduction in chromatin condensation and in contrast between condensed and noncondensed chromatin in the highly invasive C5 cells and tumor-derived cell lines. Studies on epigenetic mechanisms involving DNA methylation and/or the histone code would contribute to a better interpretation of the chromatin supraorganization changes reported herein.

DNA content and chromatin texture of human breast epithelial cells transformed with 17-beta-estradiol and the estrogen antagonist ICI 182,780 as assessed by image analysis.

The immortalized human breast epithelial MCF-10F cell line, although estrogen receptor alpha negative, develops cell proliferating activities and invasiveness indicative of neoplastic transformation, after treatment with 17-beta-estradiol (E-2). These effects are similar to those produced by benzo[a]pyrene (BP). Since we have previously reported changes in the nuclear parameters accompanying BP-induced tumorigenesis in MCF-10F cells, we have examined whether similar alterations occur in E-2-treated cells. We therefore studied DNA amounts and other nuclear parameters in Feulgen-stained MCF-10F cells after treatment with various concentrations of E-2, BP, the estrogen antagonist ICI 182,780, and E-2 in the presence of ICI 182,780. E-2 caused a certain loss of DNA and changes in the nuclear size and chromatin supraorganization of MCF-10F cells. Many of these changes were similar to those produced by BP and were indicative of neoplastic transformation. More intense chromatin remodelling was seen with 70 nM E-2. Since these changes were not abrogated totally or partially by ICI 182,780, the neoplastic transformation of MCF-10F cells stimulated by E-2 involved a process that was independent of estrogen alpha-receptors. The changes produced by ICI 182,780 alone were attributed to effects other than its well-known anti-estrogenic activity.

S-phase reduction in T47D human breast cancer epithelial cells induced by an S100P antisense-retroviral construct.

S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisense-S100P in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer. The T47D breast carcinoma cell line, a human breast epithelial cell that expresses high levels of S100P, was a tool used in this study to investigate the alteration in cell cycle induced by a retrovirus-mediated transfer of antisense-S100P. First we used the real-time PCR technique to quantify the gene expression. The results showed a reduction of 63% of expression within the T47D-S100P-A/S infected population compared with control T47D-LXSN clones. To determine the impact of the S100P antisense technique on protein expression in T47D cells, we performed immunofluorescence staining and analyzed the resulting images using a confocal microscope. The images showed much less pronounced antibody marking of the S100P protein in the T47D-S100P-A/S compared with control cells. To evaluate whether the antisense approach caused any alteration in the cell cycle, we concluded the study with flow cytometric analysis of the cell distribution. Our findings indicated that, in our model, S100P-antisense cells showed a 23% reduction of cells at the S-phase. Using transduction techniques with an S100P antisense-retroviral construct we were able to demonstrate a significant reduction in S-phase of the T47D cell cycle. To the best of our knowledge, this is the first time that an antisense approach has been used against S100P mRNA in breast cancer epithelial cells. The results showed here seem to further classify S100P as a protein that might be involved in the cell cycle imbalance observed during breast carcinogenesis.

S100P calcium-binding protein expression is associated with high-risk proliferative lesions of the breast.

Benign breast diseases represent the vast majority of diagnosis in breast pathology. However, the limited capability in identifying lesions at high risk of breast cancer evolution is an increasing problem in clinical practice. In the present study, we tested the hypothesis that the overexpression of S100P calcium-binding protein, previously identified in the very early stages of breast carcinogenesis, could be used as a marker to differentiate lesions at high risk of malignant evolution. In addition to S100P, the well-known proliferative marker, Ki-67, and estrogen receptor (ER) status were also assessed by immunohistochemistry in 155 samples from patients who submitted to stereotactic vacuum-assisted core biopsy due to breast microcalcifications. Results showed a positive association between ER and S100P overexpression, as well as a clear positive association between S100P overexpression and high-risk lesions. The strong association between S100P and ER expression highlights the hypothesis about the possible role played by S100P in the very early stages of breast carcinogenesis.

Genomic signature and susceptibility to breast cancer

Early parity is associated with a pronounced decrease in the risk of breast cancer, and additional live births reduce the risk even move. The protection afforded by early full-term pregnancy in women can be explained by the higher degree of differentiation of the mammary gland, which eliminates type 1 stem cells and creates a second type of stem cell (stem cell 2) that is able to metabolize carcinogens and repair DNA damage more efficiently than cells of the nulliparous breast. All though differentiation significantly reduces cell proliferation in the mammary gland, the epithelium remains capable of responding to a given stimulus, such as a new pregnancy. Under these circumstances, the cells that are stimulated to proliferate are derived from structures that have already been primed by the first cycle of differentiation. However, if the shift from stem cell 1 to stem cell 2 has not been completed, a sufficiently powerful carcinogenic stimulus may overburden the system, and successfully initiate a neoplastic process. Incomplete differentiation of this type may explain the development of breast cancer after a late first full-term pregnancy. The finding that differentiation is a powerful inhibitor of cancer initiation provides a strong rationale for pursuing the identification of the genes that control this process.

Cell death evaluation in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17.

The incidence of apoptosis and nuclear instability, including the incidence of catastrophic death, were investigated in benzo[a]pyrene (BP)-transformed human breast epithelial cells (BP1-E cell line) after microcell-mediated transfer of chromosomes 11 and 17. Since the introduction of normal chromosomes 11 and 17 into tumorigenic human breast BP1-E cells reverts some of these cells' characteristics (especially those affected by microsatellite instabilities and loss of heterozygosity) to those of parental non-transformed MCF-10F cells, it was expected that the cell death rates would also be affected by this treatment. The transfer of the mentioned chromosomes, especially chromosome 17, to tumorigenic BP1-E cells increased the apoptotic ratios and decreased the nuclear instability ratios, thus showing that the microsatellite instability and loss of heterozygosity induced by BP in these chromosomes of MCF-10F cells affect the control of cell death mechanisms.

DNA content, chromatin texture and nuclear morphology in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17.

BACKGROUND: A relation between the changes in DNA content and chromatin supra-organization and the expression of gradual steps of tumorigenesis has been assessed by image analysis in human breast epithelial cells (MCF-10F) treated with benzo[a]pyrene (BP) (cell lines BP1, BP1-E, BP1-Tras, and others). METHODS: Because abnormal chromosomes 11 and 17 have been associated with neoplastic progression in BP-transformed MCF-10F cells, image analysis of Feulgen-stained tumorigenic BP1-E cells with the microcell-mediated chromosome transfer of normal chromosomes 11 and 17 was carried out. RESULTS: A tendency of DNA amount distribution and nuclear size restoration to values typical of non-transformed MCF-10F cells was demonstrated, especially after the transfer of chromosome 17. No reversion in chromatin texture was found after the transfer of chromosome 11 or 17. CONCLUSIONS: Although the presence of a normal chromosome 17 should be considered among the necessary steps for tumorigenic human breast epithelial cells to recover their normality, a more complex genome balance is required for the entire nuclear chromatin of these cells to recover its totally normal supra-organization and expression.

Molecular biology and gynecology: CD 36 and its interactions with tamoxifen

Tamoxifen (TAM) is an antiestrogenic drug widely used in breast cancer treatment. By using the Differential Display technique in normal and malignant breast tissues, before and during TAM therapy, we were able to demonstrate that expression of the CD36 gene is down-regulated by this drug. CD36 is a cell-surface glycoprotein that acts as a receptor for thrombospondin-1, oxidized-LDL and collagens type I and IV. Thrombospondin-1 is involved in invasion, metastasis and angiogenesis and therefore the down-regulation of CD36 induced by TAM, might correspond to an alternative mechanism of action of this drug. CD36 is also one of the receptors for the oxidized-LDL which in turn is involved in pathogenesis of arteriosclerosis; thus the down-regulation of CD36 during TAM might explain the at least in part the lower levels of myocardial infarction during its use.

Nuclear phenotypical changes of human breast epithelial cells treated with carcinogens

Células epiteliais normales fueron obtenidas del tejido mamario de una paciente de 26 años en la cual se realizó una reducción mamoplástica. Las células fueron tratadas con los carcinógenos químicos N-nitro-N-metilurea (NMU), 4-nitroquinolina-N-oxido (NQO), o con 7,12-dimetil-benzantraceno (DMBA) en la fase logarítmica crescimento durante el primer pasaje en cultivo. Las células fueron analizadas a fin de detectar cambios nucleares fenotípicos tales como condensación de la cromatina, y cambios en el área nuclear. Tanto las células del grupo cocntrol como las tratadas exhibieron variaciones en condensación de la cromatina nuclear, que permitió clasificar los núcleos en dos tipos igualmente distribuidos: núcleos Tipo I, que eran pequeños, con cromatina finamente granular o filamentosa, y núcleos Tipo II, más grandes, con cromatina granular y un nucleolo prominente rodeado de cromatina condensada. Cada tipo nuclear fue subdividido en 5 subclases de acuerdo a variaciones en el área nuclear. El tratamiento con los carcinógenos cambió la frecuencia relativa de los tipos nucleares, con un aumento con los carcinógenos cambió la frecuencia relativa de los tipos nucleares, con un aumento de núcleos Tipo II y de aquéllos con mayor área nuclear. El tratamiento con NQO indujo el mayor cambio de frecuencia de núcleos Tipo I a Tipo II (del 50 al 80,5%), aumento del área nuclear y condensación de la cromatina. NMU y DMBA indujeron cambios similares, pero menos prominentes. Estos resultados permitieron concluir que el tratamiento in vitro de als células mamarias de esta paciente con carcinógenos químicos indujo cambios fenotípicos nucleares que podrían interpretarse como una expresión temprana de transformación celular maligna

Nuclear phenotypical changes of human breast epithelial cells treated with carcinogens

Células epiteliais normales fueron obtenidas del tejido mamario de una paciente de 26 años en la cual se realizó una reducción mamoplástica. Las células fueron tratadas con los carcinógenos químicos N-nitro-N-metilurea (NMU), 4-nitroquinolina-N-oxido (NQO), o con 7,12-dimetil-benzantraceno (DMBA) en la fase logarítmica crescimento durante el primer pasaje en cultivo. Las células fueron analizadas a fin de detectar cambios nucleares fenotípicos tales como condensación de la cromatina, y cambios en el área nuclear. Tanto las células del grupo cocntrol como las tratadas exhibieron variaciones en condensación de la cromatina nuclear, que permitió clasificar los núcleos en dos tipos igualmente distribuidos: núcleos Tipo I, que eran pequeños, con cromatina finamente granular o filamentosa, y núcleos Tipo II, más grandes, con cromatina granular y un nucleolo prominente rodeado de cromatina condensada. Cada tipo nuclear fue subdividido en 5 subclases de acuerdo a variaciones en el área nuclear. El tratamiento con los carcinógenos cambió la frecuencia relativa de los tipos nucleares, con un aumento con los carcinógenos cambió la frecuencia relativa de los tipos nucleares, con un aumento de núcleos Tipo II y de aquéllos con mayor área nuclear. El tratamiento con NQO indujo el mayor cambio de frecuencia de núcleos Tipo I a Tipo II (del 50 al 80,5%), aumento del área nuclear y condensación de la cromatina. NMU y DMBA indujeron cambios similares, pero menos prominentes. Estos resultados permitieron concluir que el tratamiento in vitro de als células mamarias de esta paciente con carcinógenos químicos indujo cambios fenotípicos nucleares que podrían interpretarse como una expresión temprana de transformación celular maligna (AU)